Activation of Calpain in Lens: A Review and Proposed Mechanism

The purpose of these experiments was to develop a hypothesis to explain activation of m-calpain in cataractogenesis observed in rodents. Thein vitromodel used to study m-calpain activation was to correlate breakdown of the ‘reporter’ protein α-crystallin with the appearance of activated m-calpain using protein sequencing and casein zymography. Incubation of α-crystallins with m-calpain and Ca2+caused proteolysis of α-crystallins and accumulation of new polypeptides. E64 and calpain inhibitor I each inhibited proteolysis of α-crystallins. TheN-terminus of the 80 kDa subunit of m-calpain was blocked at time 0 (pro calpain). After incubation with Ca2+, the remaining 80 kDa subunit of m-calpain gave aN-terminal sequence of KDREAAEGLG, indicating loss of nine amino acid from theN-terminus (autolysed calpain). The new 43 kDa m-calpain fragment also gave aN-terminal sequence of KDREAAEGLG, indicating the same loss of the first nine amino acids on theN-terminus as well as a major loss of the C-terminal half of the subunit (degraded calpain). In contrast, theN-terminus of the 80 kDa subunit of m-calpain remained blocked when E64 was present (unautolysed form). Moreover, the Ca2+concentration required for proteolysis decreased when calpain was pre-incubated with Ca2+, although proteolysis of α-crystallin required a higher Ca2+concentration than proteolysis of casein. These data suggested that the sequence of events for m-calpain activation were unautolysed, autolysed and finally degraded calpain. Unautolysed and/or autolysed calpains may be proteolytically active against α-crystallin.